Carotid Artery Injection Technique: Bounds for Bolus Mixing by Plasma and by Brain

Abstract

Estimation of Michaelis-Menten kinetic parameters ( K_m, V_max) of blood–brain barrier (BBB) transport processes with the carotid artery single injection technique assumes that mixing of the bolus with unlabeled substrate either from (a) circulating plasma or (b) amino acid efflux from brain, is minimal. The maximum extent to which the bolus could mix by these two sources is quantified in the present studies by measuring ¹⁴C-phenylalanine extraction in pentobarbital-anesthetized and conscious rats after the addition of 0–80% rat serum to the arterial injection solution. An upper bound (±SE) of bolus mixing due to mixing from both sources, expressed in terms of percentage of rat plasma, is 8.8 ± 1.9 and 7.0 ± 2.1% for the anesthetized and conscious rat, respectively. The estimated contribution to bolus mixing due to amino acid efflux from brain is 3.3 and 2.1% for the anesthetized and conscious rat, respectively. Based on these estimates, the upper bound for bolus mixing with circulating rat plasma is only 5.5 and 4.9%, respectively, for the anesthetized and conscious catheterized rat. Thus, any bolus mixing after rapid carotid injection is relatively small and is comparable to the mixing effects observed with the carotid artery infusion technique. Mixing effects on the order of 5% are shown to have no significant effect on the estimation of kinetic parameters of BBB nutrient transport, except for neutral and basic amino acid transport, which are characterized by very low K_m values relative to the usual amino acid plasma concentrations. In the rat, a 5% mixing results in an enrichment of the bolus concentration of unlabeled amino acid that approximates the K_m of the transport process, and this results in an overestimation of the absolute K_m value. However, mixing effects are shown to have little, if any, impact on the estimation of the transport V_max, K_D, or apparent K_m. Thus, amino acid influx rates predicted from kinetic constants obtained with the carotid injection technique are reliable, even if bolus mixing effects with the carotid injection technique are as high as 7–9%.